Propylisopropyl acetic acid and propylisopropyl acetamide stereoisomers, a method for their synthesis and pharmaceutical compositions containing them

ABSTRACT

The present invention relates to racemic propylisopropyl acetic acid and propylisopropyl acetamide and their isomers in their racemic and stereospecific forms, for use in treatment of neurological and psychotic disorders, and affective disorders and to treat pain, headaches and migraines. The isomers are of the compound formula I:                    
     wherein R 1  is a methyl or ethyl group; R 2  is H, methyl or an ethyl group; R 3  is ethyl or a propyl group; and R 4  is a hydroxyl or amide group; and the total number of carbon atoms in said compound is 8, provided that when R1 is a methyl group and R4 is an amide group, R2 and R3 are not ethyl, further provided that when R1 is an ethyl and R4 a hydroxyl group, only stereoisomers of the compound are referred to. The present invention further relates to a method for the stereoselective synthesis of the 2S and 2R stereoisomer of PID and PIA. The present invention also relates to pharmaceutical compositions containing as an active ingredient a racemic mixture or stereoisomers of the compounds of the general formula (I), which are useful for the treatment of neurological and psychotic disorders, and affective disorders and to treat pain, headaches and migraines.

This application is a 371 of PCT/IL99/00197 filed Apr. 12, 1999.

FIELD OF THE INVENTION

The present invention generally relates to propylisopropyl acetic acid(PIA) and propylisopropyl acetamide (PID) in their racemic andstereospecific forms, to some of their isomers and to stereoisomersthereof, for use in treatment of neurological and psychotic disorders,and affective disorders and to treat pain, including headaches andmigraine pains. The present invention further relates to a method forthe synthesis of PIA and PID stereoisomers. The present inventionfurther relates to pharmaceutical compositions containing, as an activeingredient, said racemic or stereoisomer forms.

BACKGROUND OF THE INVENTION

Headaches, especially in the form of migraine pain are a wide spreadmalady. Valproic acid (VPA), also used in antiepileptic therapy, is adrug which was approved for the treatment of migraine and has beenutilized in the treatment of epilepsy for the last 25 years with a fewside effects. Two major side effects being teratogenicity andhepatotoxicity, have been associated with valproate therapy. In humans,valpromide (VPD), which is also used as an anticonvulsant agent, is aprodrug of valproic acid (VPA). It was found to be more potent than VPAthough it exerted more significant sedative side effects (Loscher W. andNau H. (1985) Neuropharmacology 24: 427-435).

Isomers of VPD, such as valnoctamide (VCD-valmethamide or2-ethyl-3-methyl pentanamide), were found to be more potent thanvalproic acid as anticonvulsants (Haj-Yehia A. and Bialer M. (1989)Pharm Res 6:683-9). Stereoselectivity has been shown in pharmacokineticsin man for an amide of an aliphatic short-chain fatty acid such asvalnoctamide (VCD) (Barel S., Yagen B., Schurig V., Soback S., PisaniF., Perucca E. and Bialer M. (1997) Clin. Pharmacol. and Therap. 61 (4):442-449). This work demonstrated that VCD pharmacokinetics (PK) inhumans is stereoselective, with one isomer exhibiting a much higherclearance and a shorter half-life compared with the other threestereoisomers.

The present invention relates to the stereoisomers of PIA and of PID andto their isomers (such as VPD and VCA) for use in treatment ofneurological and psychotic disorders, and affective disorders and totreat pain, such as head aches. Although VPA and VPD analogues (such asVCA and VCD) were implicated in the treatment of epilepsy, there is noevidence that they in their racemic form or their individualstereoisomers are active in the treatment of neurological and psychoticdisorders, affective disorders and pain.

SUMMARY OF THE INVENTION

The present invention relates to racemic propylisopropyl acetic acid andpropylisopropyl acetamide and their isomers in their racemic andstereospecific forms, for use in treatment of neurological and psychoticdisorders, and affective disorders and to treat pain, headaches andmigraines, wherein the isomers are of the compound formula I

wherein

R₁ is a methyl or ethyl group;

R₂ is H, methyl or an ethyl group;

R₃ is ethyl or a propyl group; and

R₄ is a hydroxyl or amide group,

wherein the total number of carbon atoms in said compound is 8, providedthat when R1 is a methyl group and R4 is an amide group, R2 and R3 arenot ethyl, further provided that when R1 is an ethyl and R4 a hydroxylgroup, only stereoisomers of the compound are referred to.

The present invention further relates to a method for thestereoselective synthesis of the 2R stereoisomer of PID and PIAcomprising;

(a) synthesizing (4S)-3-(1′-oxopentyl)-4-benzyl-2-oxazolidinone from(4S)-benzyl-2-oxazolidinone (or other related oxazolidinone auxiliaries)and valeroyl chloride;

(b) synthesizing of Isopropyl trifluoromethane sulfonate (isopropyltriflate);

(c) synthesizing(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone

(d) synthesizing (2R)-propylisopropyl acetic acid ((2R-PIA) andsubsequently;

(e) synthesis of (2R)-propylisopropyl acetamide.

and to a method for the stereoselective synthesis of the 2S stereoisomerof PID and PIA comprising;

(a) Synthesizing(4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone from(4R,5S)-4-methyl-5-phenyl-2-oxazolidinone (or other relatedoxazolidinone auxiliaries) and valeroyl chloride;

(b) synthesizing(4R,5S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone;

(c) synthesizing(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone(4S,2′R)-3-(2′-isopropyl-1-oxopentyl)-4-benzyl-2-oxazolidinone;

(d) synthesizing (2S)-propylisopropyl acetic acid ((2S)-PIA)andsubsequently;

(e) synthesis of (2S)-propylisopropyl acetamide.

The present invention also relates to pharmaceutical compositionscontaining as an active ingredient a racemic mixture or stereoisomers ofthe compounds of the general formula (I), which are useful for thetreatment of neurological and psychotic disorders, and affectivedisorders and to treat pain, headaches and migraines.

BRIEF DESCRIPTION OF DRAWINGS

FIG. No. 1 shows the asymmetric synthesis of (R)-propylisopropylacetamide (R)-PID from starting material of valeroyl chloride andisopropanol through the synthesis of (4S)-benzyl, 3-(1-oxo,(2R)-isopropyl valeroyl) 2-oxazolidinone.

FIG. No. 2 shows the asymmetric synthesis of (R)-propylisopropylacetamide(R)-PID from (4S)-benzyl, 3-(1-oxo, (2R)-isopropyl valeroyl)2-oxazolidinone.

FIG. No. 3 shows the asymmetric synthesis of (S)-propylisopropylacetamide (S)-PID from (4R)-methyl-(5S)-phenyl-3-(1-oxovaleroyl)-2-oxazolidinone.

FIG. No. 4 shows the average plasma concentrations of the PIDenantiomers (R)-PID and (S)-PID for six mongrel dogs over time afteradministration of the individual enantiomers.

FIG. No. 5 shows the average plasma concentrations of the PIDenantiomers (R)-PID and (S)-PID for six mongrel dogs over time afteradministration of racemic PID.

FIG. No. 6 shows the inhibition of epoxide hydrolase by variousconcentrations of (R)-PID, (S)-PID and racemic PID.

FIG. No. 7A shows the testing regime used to test the effects of PID ondural plasma protein (bovine serum albumin (BSA)) extravasation evokedby unilateral trigeminal gangelion stimulation in anaesthetized rats.

FIG. No. 7B shows the stimulated/unstimulated versus PID mg/kg effectsof PID on dural plasma protein (bovine serum albumin (BSA))extravasation evoked by unilateral trigeminal gangelion stimulation inanaesthetized rats.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to propylisopropyl acetic acid(PIA) and propylisopropyl acetamide (PID) in their racemic andstereospecific forms, to some of their isomers and to stereoisomersthereof, for use in treatment of neurological and psychotic disorders,and affective disorders and to treat pain, including head aches andmigraine pains. The present invention further relates to the 2S and 2RPIA and PID stereoisomers and to a method for their synthesis. Thepresent invention also relates to pharmaceutical compositionscontaining, as an active ingredient, these racemic mixtures orstereoisomers.

The present invention relates to PID and PIA or PIA isomers, such asvalnoctic acid (VCA) or PID isomers, such as valpromide (VPD) in theirracemic or stereospecific forms and to the stereoisomers of chiralvalproyl amide analogous of valproic acid (VPA) such as PID, which maybe useful in the treatment of neurological and psychotic disorders, andaffective disorders and to treat pain.

The present invention relates to pharmaceutical compositions containing,as their active ingredient, PIA or PID isomers of the general formula(I):

wherein

R₁ is a methyl or ethyl group;

R₂ is H, methyl or an ethyl group;

R₃ is ethyl or a propyl group; and

R₄ is a hydroxyl or amide group,

and wherein the total number of carbon atoms is 8. provided that when R1is a methyl and R4 is an amide group, R2 is not an ethyl and R3 is notan ethyl, further provided that when R1 is an ethyl and R4 a hydroxylgroup, only stereoisomers of the compound are referred to.

For example, in PIA R1 is a methyl, R2 is a methyl, R3 is a propyl andR4 is a hydroxyl and in PID R1 is a methyl, R2 is a methyl, R3 is apropyl and R4 is an amide group.

The present invention further relates to a method for thestereoselective synthesis of the propylisopropyl acetic acid andcorresponding amide (PIA and PID)) stereoisomers (2R and 2S).

The method of synthesis of the 2R stereoisomers, which is described inFIGS. 1 and 2, comprises the following steps;

(a) synthesizing (4S)-3-(1′-oxopentyl)-4-benzyl-2-oxazolidinone (1) from(4S)-benzyl-2-oxazolidinone (or other related oxazolidinone auxiliaries)and valeroyl chloride;

(b) synthesizing of Isopropyl trifluoromethane sulfonate (isopropyltriflate) (2);

(c) synthesizing(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone(3);

(d) synthesizing (2R)-propylisopropyl acetic acid (2R-PIA) (4) andsubsequently;

(e) synthesis of (2R)-propylisopropyl acetamide (5).

The method of synthesis of the 2S stereoisomers, which is described inFIG. 3, comprises the following steps;

(a) Synthesizing(4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone (6) from(4R,5S)-4-methyl-5-phenyl-2-oxazolidinone (or other relatedoxazolidinone auxiliaries) and valeroyl chloride;

(b) synthesizing(4R,5S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone(7);

(c) synthesizing(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone

(d) synthesizing (2S)-propylisopropyl acetic acid ((2S)-PIA) (8) andsubsequently;

(e) synthesis of (2S)-propylisopropyl acetamide ((2S)-PIA) (9).

The said invention will be further illustrated by the followingexperiments. These experiments do not intend to limit the scope of theinvention, but to demonstrate and clarify it only.

The two enantiomers of PID, (2S)PID and (2R)-PID, were tested in miceand rats for their antiepileptic (anticonvulsant) activity and forneurotoxicity. Following i.p. administration to mice and oraladministration to rats, (2R)-PID was more active and showed betterpotency than (2S)-PID in the MES and sc Met tests.

In dogs, following iv administration, (2R)-PID had a lower clearance andlonger half-life than (2s)-PID, a fact that may contribute to the betteranticonvulsant activity of (2R)-PID. The better anticonvulsant activity(compare to VPA) of PID and the lack of its teratogenicity, increasesthe likelihood that other CNS activities exerted by VPA (treatment ofneurological and psychotic disorders, affective disorders pain) is morepronounced by PID in its racemic and stereoisomers forms.

The method for the asymmetric synthesis of the 2R and 2S PID and PIAstereoisomers will be further described by the following examples:

Synthesis of (2R)-Propylisopropyl Acetamide

1. (4S)-3-(1′-Oxopentyl)-4-benzyl-2-oxazolidinone

Under N₂ to a cooled solution (−78° C.) of (4S)-benzyl-2-oxazolidinone(25 g) in dry THF (150 ml) was added dropwise a solution of n-BuLi (97ml, 1.6 M in hexane). After stirring the reaction mixture for 30 min,valeroyl chloride (20.1 ml) was added dropwise via cannula, the reactionwas slowly warmed to 0° C., at this temperature stirred for 2.5 hoursand quenched by saturated NH₄CL solution. After evaporation of THF theresidue was extracted with DCM (3×150 ml). The combined organic extractswashed with water, saturated brine and dried over MgSO₄. The product(27.6 g) was crystallized from 10% EtOAc in PE, yield 75%.

4. (2R)-Propylisopropyl Acetic Acid ((2R)-PIA)

To a cooled (0° C.) solution of(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone (8.8 g)in a mixture of THF:double distilled water (DDW) (4:1, 550 ml) was addedH₂O₂ (30%, 19.3 ml) followed by a solution of LiOH (2.44 g in 50 mlDDW). After stirring for 3 hours the reaction mixture was warmed to roomtemperature (˜23° C.) and left overnight. After 24 hours the reactionmixture was cooled to 0° C. quenched by sodium sulfite (21 g in 100 mlDDW) and stirred for an additional hour. THF evaporated and the basicaqueous phase (PH=11) extracted with DCM (3×100 ml). The chiralauxiliary, (4S)-benzyl-2-oxazolidinone was obtained after evaporation ofthe DCM and crystallization from 20% EtOAc in PE, 80% yield. The aqueousphase was then acidified with concentrated HCl (PH=2) and extracted withEtOAc (3×100 ml). The combined organic extracts washed with saturatedbrine, dried on MgSO₄, evaporated and afforded the product, a colorlessoil (3.49 g), yield 83%.

3. (4S,2′R)-3-(2′-Isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone

Under N₂ to a cooled (−78° C.) solution of dry diisopropylamine (11 ml)in dry THF (40 ml) was added dropwise n-BuLi (49 ml, 1.6 M solution inhexane). After stirring the reaction mixture for 30 min a cooled (−78°C.) solution of (4S)-3-(1′-oxopentyl)-4-benzyl-2-oxazolidinone (18.5 gin 70 ml dry THF) is slowly added via cannula. After stirring for 1hour, a cooled (−78° C.) solution of isopropyl triflate (15 g in 40 mldry THF) is added via cannula. The reaction slowly warmed to −20° C.,left overnight at −20° C. and quenched by saturated NH₄Cl solution.Total reaction time was 20 hours. THF evaporated and the residueextracted with Et₂O (3×150 ml) and dried over MgSO₄. Purification of thecrude product (23 g of a yellow oil) by column chromatography (silicagel, 0.5-3% EtOAc in PE) afforded 8.8 g of a yellowish oil, 41% yield.

2. Isopropyl Trifluoromethane Sulfonate (Isopropyl Triflate)

Under N₂ to a cooled (−15° C.) solution of dry isopropanol (10.4 ml) anddry Et₃N (25.2 ml) in dry dichloromethane (DCM) (200 ml) was addeddropwise a cooled (−15° C.) solution of Tf₂O (49.0 g in 50 ml of DCM).The reaction mixture was stirred for an hour then quenched with cooled(0° C.) HCl solution (0.25 M, 2×350 ml). The organic phase was washedwith cooled (0° C.) solution of NaHCO₃ (0.5 M, 2×175 ml), dried overMgSO₄ and concentrated. The yellowish liquid obtained was dissolved in acooled (0° C.) pentane solution (50 ml), filtered through a short MgSO₄plug and concentrated. The product (18.3 g) was obtained in 55% yield,dissolved in dry pentane and kept at −20° C. until used.

5. (2R)-Propylisopropyl Acetamide ((2R)-PID)

Under N₂ to a cooled (0° C.) solution of (2R)-PIA (3.3 g) dissolved indry DCM (100 ml) and dry DMF (1.77 ml) was added dropwise a solution ofoxalyl chloride (34.3 ml, 2.0 M solution in DCM). After stirring onehour the DCM and excess oxalyl chloride were evaporated by N₂ stream. Inorder to remove traces of oxalyl chloride the crude product was treatedwith dry DCM (2×20 ml) which was evaporated by N₂ stream. To the crudereaction mixture dissolved in cooled (0° C.) dry DCM (100 ml) was addedNH₄OH (20 ml, 25% solution in water) and the reaction mixture stirredfor an hour. The organic phase washed with water and half saturatedbrine, dried over MgSO₄, filtered and concentrated. The product wascrystallized from 20% EtOAc in PE to afford 2.14 g, 65% yield.

Synthesis of (2S)-Propylisopropyl Acetamide

1. (4R,5S)-3-(1′-Oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone

(4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone wassynthesized from (4R,5S)-4-methyl-5-phenyl-2-oxazolidinone and valeroylchloride by the same procedure as(4S)-3-(1′-oxopentyl)-4-benzyl-2-oxazolidinone. The product (29.55 g)was obtained in 84% yield.

2.(4R,5S,2′S)-3-(2′-Isopropyl-1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone

(4R,5S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinonewas synthesized from(4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone and isopropyltriflate by the same procedure as(4S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone. Theproduct (5.53 g) was obtained in 32% yield.

3. (2S)-Propylisopropyl Acetic Acid ((2S)-PIA)

(2S)-PIA was synthesized from(4R,5S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinoneby the same procedure as (2R)-PIA. The product (2.33 g) was obtained in89% yield.

4. (2S)-Propylisopropyl Acetamide ((2S)-PID)

(2S)-PID was synthesized from (2S)-PIA by the same procedure as(2R)-PID. The product (1.54 g) was obtained in 67% yield.

Pharmacokinetic Studies

Pharmacokinetic experiments were carried out on six mongrel dogsweighing 18-25 kg. Dogs were housed in an animal farm and were broughtto the lab repeatedly every two-three weeks for crossover experimentsafter an overnight fast. Each dog was inserted with a urine (Levin'stube, Pennine Healthcare, Derby, UK) and two venous (20G/32 mm Venflon2, Ohmeda, Helsingborg, Sweden) catheters located on different legs.Dogs were fed with commercial dog food four hours after drug injection,and had free access to water during the whole experiment.

Each dog was intravenously injected with 70 nmole/kg (10 mg/kg) of eachenantiomer separately or 140 nmole/kg of the racemate, dissolved in 2 mlof 96% ethyl alcohol. Venous blood samples (6 ml) were withdrawn via anindwelling catheter from the other leg than used for injection, andtransferred into heparinized tubes. The blood samples were centrifugedat 3000 g for 10 minutes, plasma was then separated and stored at 20° C.until analyzed. Blood collection commenced at 5 minutes, and continuedup to 12 hours after injection. Urine samples were collected in 1-2 hourintervals beginning at 1 hour and up to 12 hours after injection. Theurine volume was recorded and an aliquot was stored at 20° C. untilanalyzed.

Assay of plasma samples: to test tubes containing 2 μg of the internalstandard (diisopropyl acetamide), 0.5 ml of plasma (thawed at roomtemperature) and 5 ml of tert-butyl methyl ether (TBME) was added. Thetest tubes were vigorously vortexed for 30 sec and centrifuged at 3000 gfor 10 minutes. The organic phase was separated and dried under reducedpressure using a vortex evaporator. Samples were then reconstituted with150 μl of chloroform and dried under reduced pressure without vortexing.The sample was again reconstituted with 30 μl of chloroform, of which 2μl were injected into the GC apparatus.

The column used for the chromatographic analysis of PID enantiomers wasa Mosandl-methyl capillary column (10 m, 0.25 mm, 0.25 μm) coated with:Heptakis (2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin asthe stationary phase and nitrogen as carrier gas (takeo carbohydr res).Column head pressure was set at 50 KPa, split ratio 1:30, oventemperature 120° C., injector temperature 250° C. and detectortemperature 250° C. At these conditions S-PID had a retention time of4.6 min, R-PID 5.1 min.

The results (FIGS. 4 and 5) show that when enantiomers are givenindividually, PID exhibits a clear stereospecific kinetics. But, whenPID is given in a racemic mixture, no sterospecifity is observed and theparmacokinetic parameters are very close to those of the individual Renantiomer.

The Pharmacokinetic parameters of the individual enantiomers and of theracemic mixture are summarized in Table 1 and Table 2 respectively.(CL=clearance, V_(β)=volume of distribution, Vss=V_(β) at steady state,MRT=Mean Residence Time, E=liver extraction ratio, fe=fraction excretedunchanged in the urine)

TABLE 1 Pharmacokinetic Parameters of PID Enantiomers in Dogs given asindividual enantiomers (10 mg/kg, via iv) (R)-PID (S)-PID S/R CL (L/h) 6.8 ± 1.6 11.3 ± 1.5 1.66* V_(β) (L) 13.1 ± 2.2 17.0 ± 2.9 1.29  Vss(L) 16.0 ± 1.2 19.1 ± 2.7 1.19  t½ (h) 1.37 ± 0.2 1.04 ± 0.1 0.76* MRT(h) 2.57 ± 0.5 1.83 ± 0.3 0.71* E (%) 15.0 ± 4.6 23.7 ± 3.5 1.58* fe (%)0.50 ± 0.4 0.40 ± 0.4 0.78* *P < 0.05.

TABLE 2 Pharmacokinetic Parameters of PID Enantiomers in Dogs given in aracemic mixture (20 mg/kg, via iv) (R)-PID (S)-PID S/R CL (L/h)  6.0 ±1.7  6.0 ± 1.8 1.0 V_(b) (L) 10.2 ± 0.9 10.4 ± 1.4 1.0 Vss (L) 15.7 ±1.5 15.7 ± 0.9 1.0 t½ (h) 1.25 ± 0.3 1.27 ± 0.3 1.0 MRT (h) 2.83 ± 0.62.87 ± 0.7 1.0 E (%) 12.5 ± 4.0 12.1 ± 4.1 1.0 fe (%)  0.60 ± 0.45  0.66± 0.46 1.1

Epoxide Hydrolase Inhibition Assay

PID is known to be an inhibitor for Epoxide Hydrolase (EH) activity. Astudy was conducted in order to explore the possibility of PIDstereospecificy in the EH inhibition. The study was conducted asfollows:

The in vitro microsomal EH inhibitory potency of racemic PID and itsindividual enantiomers was measured in human liver microsomes withS-(+)-styrene oxide (SO) as substrate. Microsomes were prepared fromhuman liver #135 (HL-135), genotipically classified as a wild type EH,according to the previously published procedure (rettie, hasset). Therate of S-(+)-1-phenyl-1,2-ethanediol (PED) formation was measured inmicrosomal incubations, as previously described, with slightmodifications (kerr cpt 89). Briefly: Racemic PID, S-PID, R-PID or 0.1Msodium phosphate buffer PH 7.4 were preincubated with microsomal proteinfor 1.5 min at 37° C., before the reaction was started by addition ofSO. The reaction was terminated by the addition of 3 ml n-hexane, rapidvortexing for 30 sec, and placing the tubes on ice until processedfurther. The background hydrolysis rate for SO was measured byreplacement of microsomal protein with an equal amount of denaturedmicrosomal protein. All reported PED formation rates have been correctedfor the non-enzymatic hydrolysis. The final protein concentration was5.34 μg/ml. SO was added in a 15 μl acetonitrile solution, such that thefinal SO concentration was 25 μM (equals the K_(m) of SO). PID and itsindividual enantiomers were added in a 15 μl methanolic solution, suchthat the final concentration of organic solvent was 1%. The inhibitionreactions were investigated at five concentrations ranging from 4 to 30μM. Positive control for 50% inhibition was done by using 5 μM VPD(equals the IC₅₀ of VPD). All determinations were made in triplicates.

PED was extracted and assayed using a reverse phase HPLC procedurepreviously published, with slight modifications (kerr cpt 89). Internalstandard was felbamate 1.6 μg in 100 μl methanolic solution. Microsomalincubations were extracted with 7 ml TBME. Mobile phase composition:double distilled water/acetonitrile/methanol 70:20:10. Flow rate was setat 1.2 ml/min and the compounds detected by UV absorption at 210 mn.Chromatographic separation was carried out on a Zorbax C₈ column (5 μM,4.6×25 cm) equipped with a C₈ guard column (5 μM, 4.6×1.0 cm).

The inhibition of EH mediated SO hydrolysis by PID and the individualenantiomers; S-PID and R-PID, was examined in microsomal suspensionsthat were prepared from a single human liver, HL-135. The PED formationrates are presented inn FIG. 6. From percent remaining activity of EHvs. inhibitor concentration plots, IC₅₀ values could be derived for eachof the tested compounds. Racemic PID had an IC50 of 8.55 μM, S-PID anIC₅₀ of 7.60 μM and R-PID an IC₅₀ of 11.20 μM.

Average non-enzymatic SO hydrolysis rates were 6.67±0.47% of the controlenzymatic hydrolysis rates. VPD (5 μM) as a positive control inhibitedSO hydrolysis by 51.5±2.4% of the control enzymatic hydrolysis rates. QCof four different PED concentrations within the PED concentration rangehad an accuracy of 0.55-2.70% and reproducibility (%CV) of 1.49-5.61%.

Biological Activity

1. Teratogenicity Study

1a. Teratogenicity (Finnel) Study in SWV Mice

Teratogenicity was evaluated in the highly inbred SWV mice strain on thebasis of their known susceptibility to VPA-induced NTDs (finnelteratology 88). The mice were maintained on a 12 h light cycle in theAnimal Resources Facility at The college of Veterinary Medicine, TexasA&M University. Mice were pathogen free and were allowed free access toWayne TekLad rodent chow and tap water. Virgin females, 40-60 days ofage were bred overnight and examined the next morning for the presenceof vaginal plugs. The beginning of gestation (day 0) was set at 10 P.M.of the previous evening, the midpoint of the dark cycle (snell 48). Tendams were randomly assigned to each of the tested compounds; racemicPID, S-PID and R-PID. At day 8.5 of gestation, each dam was exposed to asingle intraperitoneal (ip) injection of the tested compound (500-600mg/kg) or the vehicle (1% carboxymethyl cellulose-CMC). Followingadministration, the dams were returned to their cages until day 18.5 ofgestation. At that time the dams were sacrificed by cervicaldislocation, the abdomen opened and the uterine contents removed. Thelocation of all-viable embryos or fetuses and resorption sites wererecorded, and the embryos were examined for the presence of exencephaly.

Teratogenicity in the SWV mice strain was evaluated following a singleip administration of PID, S-PID and R-PID to ten dams at day 8.5 ofgestation. Teratogenic data is presented in Table 1. Both PID and R-PIDwere administered at doses of 600 mg/kg. Due to several incidences ofmaternal lethality following 600 mg/kg administration of S-PID,teratogenicity was further investigated at 500 mg/kg. Both racemic andR-PID failed to induce exencephaly in the SWV embryos. S-PID caused 0.8%exencephaly, but this was not different from controls (0%). Resorptionrates induced by PID, S-PID and R-PID were 6.3%, 6.1% and 10.4%,respectively, which is not different from controls, 8.6%.

TABLE 3 Teratogenic effects of PID, R-PID and S-PID in SWV mice CompoundDose (mg/kg) Litters Implants¹ Resorptio Live Fetuses² ExencephalControl³ 0 11 148 1 (0.7) 147 0 PID 600 10 126 8 (6.3)* 118 0 R-PID 60010 134 14 (10.4) 120 0 S-PID 500 10 131 8 (6.1)* 123 1 (0.8) ¹Percent oftotal implants ²Percent of live fetuses ³Controls were adininisteredwith the vehicle: CMC 1% *Significantly different relative to controls(P < 0.05)

1b. Teratogenicity (Nau) Study in Mice of NMRI Strain

Mice of the NMRI strain (Harlan-Winkelmann GmbH, 33176 Borchen, Germany)were kept under controlled conditions: Room temperature (21±1° C.),relative humidity (50±5%), and a 12 hour light-dark cycle with the lightperiod from 10 a.m. to 10 p.m. Females weighing 28 to 36 g were matedwith males of the same strain for 3 hours (from 6 a.m. to 9 a.m.).Animals with vaginal plugs were separated, and the following 24 hourperiod was designated as day 0 of pregnancy. The animals were given freeaccess to food (Altromin 1324 diet, Lage, Germany) and tap water.Approval for the study was obtained from the Department of Health.

Sodium valproate (VPA-Na), racemic PID, (2R)-PID and (2S)-PID weresuspended in a 25% Cremophor EL aqueous solution. For another treatmentgroup, VPA-Na was dissolved in distilled water. The pregnant dams wereinjected with a single 3 mmol/kg subcutaneously dose (10 ml/kg volumeadministered) on the morning of day 8 of gestation. Mice of the controlgroup were injected with the vehicle, 25% Cremophor EL solution (10ml/kg volume administered). On day 18 of gestation the darns weresacrificed by cervical dislocation, the uteri removed and the number ofimplantations, resorptions and dead fetuses recorded. Living fetuseswere weighed individually and inspected for the presence of externalmalformations.

Teratogenic potency of racemic PID and the individual enantiomers wasevaluated in NMRI mice following a single 3 mmol/kg subcutaneousinjection to pregnant dams at day 8 of gestation, as presented in Table4. Racemic PID and the individual enantiomers failed to induceexencephaly in the developing mouse embryos, whereas VPA caused 37% and73% exencephaly in living fetuses when administered in aqueous solutionand Cremophor suspension, respectively. Fetal deaths and earlyresorptions expressed as embryolethality was significantly increased inVPA-treated animals, whereas all PID groups and controls had comparablerates. All treatment groups (PID and VPA) had significant reduction infetal weight.

TABLE 4 Teratogenicity of racemic PID, (2R)-PID and (2S)-PID in NMR1mice at day 18 of gestation. Dose Litters Total Implants Live FetusesFetal Weight¹ Embryolethality² Exencephaly³ ompound mmol/kg n n n g n(%) n (%) (2R)-PID4 3 9 92 78 1.16 ± 0.08* 14 (15.2) 0 (0) (2S)-PID4 3 564 58 1.15 ± 0.09* 6 (9.4) 0 (0) Racemic PID4 3 8 96 87 1.21 ± 0.07  9(9.4) 0 (0) VPA-Na4 3 7 86 41 0.96 ± 0.08* 45 (52.3)** 30 (73.2)**VPA-Na5 3 8 92 63 1.09 ± 0.11* 29 (31.5)** 23 (36.5)** Controls — 20 258238 1.23 ± 0.09  20 (7.8) 3 (1.3) ²Percent of total implants ³Percent oflive fetuses ⁴Administered subcutaneously in the vehicle: 25% CremophorEL. ⁵Administered intraperitonealy dissolved in distilled water.⁶Controls received the vehicle: 25% Cremophor EL. *Significantlydifferent from controls (p < 0.0001, Fisher's Exact test)**Significantly different from controls (p < 0.0001, Student's t-test)

2. Anti-Migraine Activity of PID.

The GABA transaminase inhibitor and activator, of glutamic acid, thedecarboxylase, valproic acid is being used for the treatment ofmigraine. In this study, a valproyl amide analog (PID) in a racemicform, was tested in animal models (rats) for the treatments of migraineand pain, in comparison to VPA.

The animal model used in this study is the one developed by Moskowitz etal. (F. M. Cutrer, V. Limmroth and M. A. Moskowitz: Possible mechanismsof valproate in migraine prophylaxis, Cephalalgia 17:93-100 (1997)).Moskowitz et. al. examined the plasma protein extravasation followingelectrical trigeminal ganglion stimulation or intravenous administrationof substance P. It was concluded that in this model valproic acid blocksplasma extravasation in the meninges through GABA_(A)-mediatedpostjunctional receptors probably within the meninges. The dosagesrequired are comparable to those used clinically. Thus, agonists andmodulators at the GABA_(A) receptor may become useful for thedevelopment of selective drugs for migraine and cluster headache (W. S.Lee et. al.: Peripheral GABAA receptor mediated effects of sodiumvalproate on durnal plasma protein extravasation to substance P andtrigeminal stimulation, Toward Migraine 2000, F. C. Rose Ed. Elsevier,Amsterdam, 1996, pp.289-319).

We tested the effects of PID on dural plasma protein (Bovine SerumAlbumine—BSA) extravasation evoked by unilateral trigeminal gangelionstimulation in anaesthetized rats. The results of this study, shown inFIG. 7, show that PID has inhibitory effects on the dural plasma proteinextravasation, i.e., it has the potency for anti-migraine and anti-painactivity.

3. Anticonvulsant Activity and Neurotoxicity of PID in Mice

Individual enantiomers of PID were screened in mice for theiranticonvulsant activity (by the NIH Epilepsy Branch) followingintraperitoneal administration to mice by employing a screeningprocedure which involves: (i) the maximal electoshock (MES) test, whichmeasures seizure spread; (ii) the subcutaneous pentylenetetrazol testsc. Met. Test), which measures seizure threshold; and (iii) the rotorodataxia test, which assesses neurotoxicity.

Table 6 shows the results obtained in this study:

TABLE 6 Anticonvulsant Activity and Neurotoxicity of PID in Mice(intraperitoneal administration) PID (S)-PID (R)-PID S/R MES 122 145 1101.32 sc Met 77 80 67 1.19 Neurotox. <120 118 <145 >0.81 PI-MES <0.980.81 <1.3 PI-sc Met <1.56 1.46 <2.2

4. Anticonvulsant Activity and Neurotoxicity of PID in Rats

Individual enantiomers of PID were screened in rats for theiranticonvulsant activity following oral feeding, by the proceduredescribed in the previous section. The ED₅₀ values obtained in thisstudy are as follows:

ED₅₀ for (2R)-PID: 16 mg/kg

ED₅₀ for (2S)-PID: 26 mg/kg

ED₅₀ for racemate: 22 mg/kg.

What is claimed is:
 1. A method for the stereoselective synthesis of the2R stereoisomer of propylisopropyl acetic acid and propylisopropylacetamide comprising; (a) synthesizing(4S)-3-(1′-oxopentyl)-4-benzyl-2-oxazolidinone from(4S)-benzyl-2-oxazolidinone, or other oxazolidinone auxiliaries, andvaleroyl chloride; (b) synthesizing of Isopropyl trifluoromethanesulfonate (isopropyl triflate); (c) synthesizing(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone (d)synthesizing (2R)-propylisopropyl acetic acid (2R-PIA) and subsequently;(e) synthesis of (2R)-propylisopropyl acetamide (2R-PID).
 2. A methodfor the stereoselective synthesis of the 2S stereoisomer ofpropylisopropyl acetic acid and propylisopropyl acetamide comprising;(a) synthesizing(4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone from(4R,5S)-4-methyl-5-phenyl-2-oxazolidinone, or other oxazolidinoneauxiliaries, and valeroyl chloride; (b)synthesizing(4R,5S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone;(c) synthesizing(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone; (d)synthesizing (2S)-propylisopropyl acetic acid (2S-PIA) and subsequently;(e) synthesis of (2S)-propylisopropyl acetamide (2S-PID).
 3. A methodfor the stereoselective synthesis of the 2R stereoisomer ofpropylisopropyl acetic acid and propylisopropyl acetamide according toclaim 1 wherein step (a) comprises adding n-BuLi to a solution of(4S)-benzyl-2-oxazolidinone at approximately −78° C., stirring andadding valeroyl chloride, warming to approximately 0° C., stirring andquenching obtaining (4S)-benzyl,3-(1-oxo valeroyl) 2-oxazolidinone(compound (1)), and wherein step (b) comprises adding to a solution ofisopropanol and Et3N a solution of triflic anhydride at approximately−15° C., stirring, quenching, obtaining isopropyl triflate (compound(2)) in an organic phase; and wherein step (c) comprises adding n-BuLito a solution of diisopropylamine oxazolidinone at approximately −78°C., stirring and adding a solution of(4S)-3-(1′-oxopentyl)-4-benzyl-2-oxazolidinone, stirring and adding asolution of isopropyl triflate, warming the solution to −20° C. andquenching obtaining (4S)-benzyl, 3-(1-oxo, (2R)-isopropyl valeroyl)2-oxazolidinone (compound (3)); and wherein step (d) comprises addingH₂O₂ followed by a solution of LiOH to compound (3), stirring andwarming to about 23° C., cooling to 0° C. and quenching obtaining abasic aqueous phase, further extracting with dichloromethane obtaining(4S)-benzyl-2-oxazolidinone, further acidifying said(4S)-benzyl-2-oxazolidinone obtaining (R)-propylisopropyl acetic acid(compound (4)); and wherein step (e) comprises adding a solution ofoxalyl chloride to a solution of compound (4) at approximately 0° C.,stirring and adding NH₄OH, stirring and obtaining (R)-propylisopropylacetamide (compound (5)) in an organic phase.
 4. A method for thestereoselective synthesis of the 2S stereoisomer of propylisopropylacetic acid and propylisopropyl acetamide according to claim 2 whereinstep (a) comprises adding n-BuLi to a solution of(4R,5S)-4-methyl-5-phenyl-2-oxazolidinone) at approximately −78° C.,stirring and adding valeroyl chloride, warming to 0° C., stirring andquenching obtaining (4R)-methyl-(5S)-phenyl-3-(1-oxovaleroyl)-2-oxazolidinone (compound (6)); and wherein step (b) comprisesadding to a cooled solution of isopropanol and Et3N a solution oftriflic anhydride at approximately −15° C., stirring, quenching,obtaining isopropyl triflate (compound (2)) in an organic phase; andstep (c) comprises adding n-BuLi to a solution of diisopropylamine atapproximately −78° C., stirring and adding a solution of (4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone, stirring andadding a solution of isopropyl triflate, warming the solution to −20° C.and quenching obtaining (4R)-methyl-(5S)-phenyl-3-(1-oxo-(2S)-isopropylvaleroyl) 2-oxazolidinone (compound (7)); and wherein step (d) comprisesadding H₂O₂ followed by a solution of LiOH to compound (7), stirring andwarming to about 23° C., cooling to 0° C. and quenching obtaining abasic aqueous phase, further extracting with dichloromethane, acidifyingsaid extract obtaining (S)-propylisopropyl acetic acid (compound (8));and wherein step (e) comprises adding a solution of oxalyl chloride to asolution of (R)-propylisopropyl acetic acid (compound (4)) atapproximately 0° C., stirring and adding NH₄OH, stirring and obtaining(S)-propylisopropyl acetamide (compound (9)) in an organic phase.